PROJECT SUMMARY/ABSTRACT Eosinophilic esophagitis (EoE) is a rapidly emerging chronic disease, predominantly triggered by food antigens, that progresses from inflammation to fibrosis. Despite the name eosinophilic esophagitis, increasing evidence suggests that mast cells (MC), which drive fibrosis in conditions such as asthma and scleroderma, are integral to the pathogenesis of EoE. MCs classically release histamine and other inflammatory mediators upon cross-linking of immunoglobulin E (IgE), but multiple lines of evidence have demonstrated that EoE is not mediated by IgE. MCs can also be activated by a range of cationic substances, including opiates, through a novel G-protein coupled receptor, MAS-related G protein-coupled receptor X2 (MRGPRX2). Milk and wheat are the two most common triggers of EoE, and digestion of these foods produces homologous 7 amino acid (AA) peptides, beta-casomorphin (BCM7) and gliadorphin (G7), respectively, that can engage opioid receptors and activate rat peritoneal MCs. Our group recently found that these two peptides also activate the human LUVA MC line. Using a HEK293 MRGPRX2 transfected cell line, our group further found that the opiate-like peptides BCM7 and G7 can activate these cells only in the presence of MRGPRX2. This finding leads to our central hypothesis that MCs are integral to the pathogenesis of EoE, and that non-IgE mediated activation of MCs occurs through food-induced activation of MRGPRX2. Dr. McGowan proposes to explore this hypothesis by examining 1) whether BCM7 and G7 activate MCs through MRGPRX2 using HEK293 cells with and without stably transfected MRGPRX2 and LUVA cells with and without siRNA-mediated silencing of MRGPRX2; 2) whether BCM7 and G7 stimulation of MCs leads to the production of mediators that are known to contribute to the fibrosis and eosinophil recruitment seen in EoE; and 3) whether esophageal MCs in patients with EoE express MRGPRX2 and correlate with fibrotic features. Elucidating the mechanism by which BCM7 and G7 activate MCs and lead to the inflammation and fibrosis seen in EoE will have broad implications for our understanding of EoE pathogenesis, as well as other food- related conditions such as irritable bowel syndrome and non-celiac gluten sensitivity that are also triggered by milk and wheat ingestion. This is a question with significant public health implications, as BCM7 and G7 are widely consumed in the United States and other westernized countries, where the prevalence of EoE is increasing. Demonstrating activation of MCs by these food-derived peptides could lead to a paradigm shift in the way that we view the pathogenesis and treatment options for this disease.